溶栓药APSAC的制备和鉴定

PREPARATION AND IDENTIFICATION OFANISOYLATED PLASMINOGEN STREPTOKINASE ACTIVATOR COMPLEX

重组链激酶（ｒ－ＳＫ）由工程菌提取液经ＳｅｐｈａｃｒｙｌＳ－２００柱纯化，比活性达１０５ＩＵ／ｍｇ，十二烷基硫酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）显示分子量为７８．０２４７×１０－２４ｋｇ的一条带。人新鲜血浆经Ｌｙｓｉｎｅ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析，ＳｅｐｈａｄｅｘＧ－２５凝胶过滤制备纤溶酶原（ｐｌｇ），比活性达２３．７ＩＵ／ｍｇ，活性回收率为１３．６％，ＳＤＳ－ＰＡＧＥ显示分子量为１４７．７４８９×１０－２４ｋｇ的一条带。对－茴香酸－对－脒基ｒ－ＳＫ和ｐｌｇ的混合物经苯酯盐酸盐（ＡＰＡＮ）修饰后制备茴香酰纤溶酶原链激酶激活剂复合物（ＡＰＳＡＣ），产物经Ｌ－ｌｙｓｉｎｅＳｅｐｈａｒｏｓｅ４Ｂ亲和层析，ＳＤＳ－ＰＡＧＥ显示分子量为７８．０２４７×１０－２４ｋｇ和１４７．７４８９×１０－２４ｋｇ两条带，其拟一级水解速率常数为１．２６×１０－４ｓｅｃ－１。体外溶栓实验证明其有溶栓活性。

Recombinant streptokinase (r-SK)was purified by sephacryl S-200 gel filtration.The specific activity of the final product was 105 IU/mg and the MW was 78.0247×10-24kg.Human plasminogen was purified from human fresh plasma by L-lysine-sepharose 4B and sephadex G-25 gel filtration. The specific activity,recovery and molecular weight were 23.7IU/mg,13.67% and 147.748 9 ×10-24kg respectively.r-SK and plasminogen were mixed,modified by p-Amidinophenyl-p'-Anisate.HCl(APAN) and applied to a L-lysine-sepharose 4B collumn.The final product was analysed by SDS-PAGE.There were two bands corresponding to plasminogen(MW,147.748 9×10-24kg)and r-SK(MW,1478.024 7×10-24 kg)The pseudo-first order rate constant was 1.26×10-4 sec-1 measured by chromagenic substrate S2390 The fibrinolytic effect was proved by the fibrinolytic test in vitro.